Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A human leukocyte antigen (HLA)-matched unrelated bone marrow transplantation (BMT) was performed in a 13-year-old patient with the congenital immunodeficiency syndrome, Wiskott-Aldrich syndrome. The patient had a history of acyclovir (ACV)-resistant (ACV(r)) herpes simplex virus type 1 (HSV-1) infections prior to BMT. After BMT, the skin lesions caused by HSV-1 relapsed on the face and genito-anal areas. Ganciclovir (GCV) therapy was initiated, but the mucocutaneous lesions worsened. An HSV-1 isolate recovered from the lesions during this episode was resistant to both ACV and GCV. The ACV(r) isolate was confirmed to have the same mutation in the viral thymidine kinase (TK) gene as that of the previously isolated ACV(r) isolates from the patient. After treatment switch to foscarnet (PFA), there was a satisfactory remission but not a complete recovery. Although the mucocutaneous lesions improved, a PFA-resistant (PFA(r)) HSV-1 was isolated 1 month after the start of PFA therapy. The PFA(r) HSV-1 isolate coded for the same mutation in the viral TK gene as the ACV(r) HSV-1 isolates. Furthermore, the PFA(r) isolate also expressed a mutated viral DNA polymerase (DNA pol) with an amino acid (Gly) substitution for Val at position 715. This is the first report on the clinical course of a BMT-associated ACV(r) HSV-1 infection that subsequently developed resistance to foscarnet as well.
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PMID:Bone marrow transplantation in a child with Wiskott-Aldrich syndrome latently infected with acyclovir-resistant (ACV(r)) herpes simplex virus type 1: emergence of foscarnet-resistant virus originating from the ACV(r) virus. 1221 Apr 36

DNA polymerase was purified from soybean (Glycine max) chloroplasts that were actively replicating DNA. The main form (form I) of the enzyme was associated with a low level of 3[prime] to 5[prime] exonuclease activity throughout purification, although the ratio of exonuclease to polymerase activity decreased with each successive purification step. A second form (form II) of DNA polymerase, which elutes from DEAE-cellulose at a higher salt concentration than form I, was devoid of any exonuclease activity. To assess the potential function of the 3[prime] to 5[prime] exonuclease in proofreading, the fidelity of deoxynucleotide incorporation was measured for form I DNA polymerase throughout purification. Despite the steadily decreasing ratio of 3[prime] to 5[prime] exonuclease to polymerase activity, the extent of misincorporation by form I enzyme remained unchanged during the final purification steps, suggesting that the exonuclease did not contribute to the accuracy of DNA synthesis by this polymerase. Fidelity of form I DNA polymerase, when compared with that of form II, revealed a higher level of misincorporation for form I enzyme, a finding that is consistent with the exonuclease playing little or no role in exonucleolytic proofreading.
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PMID:Accuracy of Deoxynucleotide Incorporation by Soybean Chloroplast DNA Polymerases Is Independent of the Presence of a 3[prime] to 5[prime] Exonuclease. 1222 34

Structural differences between class A and B DNA polymerases suggest that the motif B region, a wall of the catalytic pocket, may have evolved differentially in the two polymerase families. This study examines the function of the motif B residues in Saccharomyces cerevisiae DNA polymerase alpha (pol alpha). Effects of the mutations were determined by biochemical analysis and genetic complementation of a yeast strain carrying a temperature-sensitive pol alpha mutant. Many conserved residues were viable with a variety of substitutions. Among them, mutations at Asn-948 or Tyr-951 conferred up to 8-fold higher colony formation frequency in a URA3 forward mutation assay, and 79-fold higher trp1 reversion frequency was observed for Y951P in yeast. Purified Y951P was as accurate as wild type in DNA synthesis but approximately 6-fold less processive and 22-fold less active in vitro. Therefore, Y951P may increase the frequency of mutant colony formation because of its low level of DNA polymerase activity in yeast. Mutations at Lys-944 or Gly-952 were not viable, which is consistent with the observation that mutants with substitutions at Gly-952 have strongly reduced catalytic activity in vitro. Gly-952 may provide a space for the nascent base pair and thus may play an essential function in S. cerevisiae DNA pol alpha. These results suggest that class B DNA polymerases have a unique structure in the catalytic pocket, which is distinct from the corresponding region in class A DNA polymerases.
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PMID:Distinct function of conserved amino acids in the fingers of Saccharomyces cerevisiae DNA polymerase alpha. 1263 57

Gly-952 is a conserved residue in Saccharomyces cerevisiae DNA polymerase alpha (pol alpha) that is strictly required for catalytic activity and for genetic complementation of a pol alpha-deficient yeast strain. This study analyzes the role of Gly-952 by characterizing the biochemical properties of Gly-952 mutants. Analysis of the nucleotide incorporation specificity of pol alpha G952A showed that this mutant incorporates nucleotides with extraordinarily low fidelity. In a steady-state kinetic assay to measure nucleotide misincorporation, pol alpha G952A incorporated incorrect nucleotides more efficiently than correct nucleotides opposite template C, G, and T. The fidelity of the G952A mutant polymerase was highest at template A, where the ratio of incorporation of dCMP to dTMP was as high as 0.37. Correct nucleotide insertion was 500- to 3500-fold lower for G952A than for wild type pol alpha, with up to 22-fold increase in pyrimidine misincorporation. The Km for G952A pol alpha bound to mismatched termini T:T, T:C, C:A, and A:C was 71- to 460-fold lower than to a matched terminus. Furthermore, pol alpha G952A preferentially incorporated pyrimidine instead of dAMP opposite an abasic site, cis-syn cyclobutane di-thymine, or (6-4) di-thymine photoproduct. These data demonstrate that Gly-952 is a critical residue for catalytic efficiency and error prevention in S. cerevisiae pol alpha.
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PMID:The Gly-952 residue of Saccharomyces cerevisiae DNA polymerase alpha is important in discriminating correct deoxyribonucleotides from incorrect ones. 1263 58

DNA polymerase (pol) lambda is homologous to pol beta and has intrinsic polymerase and terminal transferase activities. However, nothing is known about the amino acid residues involved in these activities. In order to precisely define the nucleotide-binding site of human pol lambda, we have mutagenised two amino acids, Tyr505 and the neighbouring Phe506, which were predicted by structural homology modelling to correspond to the Tyr271 and Phe272 residues of pol beta, which are involved in nucleotide binding. Our analysis demonstrated that pol lambda Phe506Arg/Gly mutants possess very low polymerase and terminal transferase activities as well as greatly reduced abilities for processive DNA synthesis and for carrying on translesion synthesis past an abasic site. The Tyr505Ala mutant, on the other hand, showed an altered nucleotide binding selectivity to perform the terminal transferase activity. Our results suggest the existence of a common nucleotide-binding site for the polymerase and terminal transferase activities of pol lambda, as well as distinct roles of the amino acids Tyr505 and Phe506 in these two catalytic functions.
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PMID:Mutagenesis of human DNA polymerase lambda: essential roles of Tyr505 and Phe506 for both DNA polymerase and terminal transferase activities. 1462 24

Escherichia coli cells expressing the mutA allele of a glyV (glycine tRNA) gene express a strong mutator phenotype. The mutA allele differs from the wild type glyV gene by a base substitution in the anticodon such that the resulting tRNA misreads certain aspartate codons as glycine, resulting in random, low-level Asp-->Gly substitutions in proteins. Subsequent work showed that many types of mistranslation can lead to a very similar phenotype, named TSM for translational stress-induced mutagenesis. Here, we have determined the specificity of forward mutations occurring in the lacI gene in mutA cells as well as in wild type cells. Our results show that in comparison to wild type cells, base substitutions are elevated 23-fold in mutA cells, as against a eight-fold increase in insertions and a five-fold increase in deletions. Among base substitutions, transitions are elevated 13-fold, with both G:C-->A:T and A:T-->G:C mutations showing roughly similar increases. Transversions are elevated 35-fold, with G:C-->T:A, G:C-->C:G and A:T-->C:G elevated 28-, 13- and 27-fold, respectively. A:T-->T:A mutations increase a striking 348-fold over parental cells, with most occurring at two hotspot sequences that share the G:C-rich sequence 5'-CCGCGTGG. The increase in transversion mutations is similar to that observed in cells defective for dnaQ, the gene encoding the proofreading function of DNA polymerase III. In particular, the relative proportions and sites of occurrence of A:T-->T:A transversions are similar in mutA and mutD5 (an allele of dnaQ) cells. Interestingly, transversions are also the predominant base substitutions induced in dnaE173 cells in which a missense mutation in the alpha subunit of polymerase III abolishes proofreading without affecting the 3'-->5' exonuclease activity of the epsilon subunit.
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PMID:Specificity of spontaneous mutations induced in mutA mutator cells. 1506 31

We reported previously that a novel dipeptide alcohol, l-homoserylaminoethanol (Hse-Gly-ol), is a selective inhibitor of eukaryotic DNA polymerase epsilon (pol epsilon) [Bioorg. Med. Chem.2004, 12, 957-962]. The discovery suggests that the dipeptide structure could be a chemical frame for a DNA polymerase inhibitor. Therefore, we chemically synthesized 27 different species of dipeptide alcohols, and tested this inhibitory capability. Compound 6 (l-aspartylaminoethanol, Asp-Gly-ol) was found to be the strongest pol alpha inhibitor. Compound 6 did not influence the activities of other replicative DNA polymerases such as delta and epsilon, and had no effect on the activities of prokaryotic DNA polymerases, nor DNA metabolic enzymes such as human immunodeficiency virus type 1 reverse transcriptase, T7 RNA polymerase and bovine deoxyribonuclease I. The inhibitory effect of compound 6 on pol alpha was dose-dependent, and 50% inhibition was observed at a concentration of 33.5 microM. Compound 6-induced inhibition of pol alpha activity was non-competitive with both the DNA template-primer and the dNTP substrate. This is the first report on a water-soluble pol alpha-specific inhibitor, sought for precise biochemical studies of pol alpha. The relationships between the structures of dipeptide alcohols and the inhibition of eukaryotic DNA polymerases are discussed.
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PMID:Dipeptide alcohol-based inhibitors of eukaryotic DNA polymerase alpha. 1572 71

We reported previously that a novel dipeptide alcohol, L-homoserylaminoethanol (Hse-Gly-ol), is a selective inhibitor of eukaryotic DNA polymerase epsilon (pol epsilon). The discovery suggests that the dipeptide structure could be a chemical frame for a DNA polymerase inhibitor. Therefore, we chemically synthesized 14 different species of dipeptide alcohols and their derivatives, and tested this inhibitory capability. The mercapto group in the dipeptide alcohol was found to be important, and compound 4 (L-cysteinylaminoethanol, Cys-Gly-ol) was the strongest pol alpha inhibitor. Compound 4 did not influence the activities of other replicative DNA polymerases such as delta and epsilon, and had no effect on the activities of prokaryotic DNA polymerases, nor DNA metabolic enzymes such as human immunodeficiency virus type-1 reverse transcriptase, T7 RNA polymerase and bovine deoxyribonuclease I. The inhibitory effect of compound 4 on pol alpha was dose-dependent, and 50% inhibition was observed at a concentration of 14.8 microM. Compound 4-induced inhibition of pol alpha activity was non-competitive with both the DNA template-primer and the nucleotide substrate. The relationships between the structures of dipeptide alcohol and the inhibition of eukaryotic DNA polymerases are discussed.
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PMID:Inhibitory effect of dipeptide alcohol derivatives containing mercapto group on eukaryotic DNA polymerase alpha. 1614

Eukaryotic DNA polymerase (Pol) delta replicates chromosomal DNA and is also involved in DNA repair and genetic recombination. Motif A in Pol delta, containing the sequence DXXXLYPSI, includes a catalytically essential aspartic acid as well as other conserved residues of unknown function. Here, we used site-directed mutagenesis to create all 19 amino acid substitutions for the conserved Leu(612) in Motif A of Saccharomyces cerevisiae Pol delta. We show that substitutions at Leu(612) differentially affect viability, sensitivity to genotoxic agents, cell cycle progression, and replication fidelity. The eight viable mutants contained Ile, Val, Thr, Met, Phe, Lys, Asn, or Gly substitutions. Individual substitutions varied greatly in the nature and extent of attendant phenotypic deficiencies, exhibiting mutation rates that ranged from near wild type to a 37-fold increase. The L612M mutant exhibited a 7-fold elevation of mutation rate but essentially no detectable effects on other phenotypes monitored; the L612T mutant showed a nearly wild type mutation rate together with marked hypersensitivity to genotoxic agents; and the L612G and L612N strains exhibited relatively high mutation rates and severe deficits overall. We compare our results with those for homologous substitutions in prokaryotic and eukaryotic DNA polymerases and discuss the implications of our findings for the role of Leu(612) in replication fidelity.
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PMID:Mutator phenotypes caused by substitution at a conserved motif A residue in eukaryotic DNA polymerase delta. 1634 51

Chromatin isolated from soybean (Glycine max L., var. Wayne) hypocotyls was capable of catalyzing the polymerization of labeled deoxyribonucleoside triphosphate in the presence of the three other deoxyribonucleoside triphosphates into a trichloroacetic acid-insoluble product. This product was insensitive to base hydrolysis and ribonuclease, but was sensitive to acid hydrolysis and deoxyribonuclease. Chromatin-DNA polymerase required Mg(2+) and all four deoxyribonucleoside triphosphates for maximal activity. Inorganic pyrophosphate and actinomycin D inhibited the polymerase activity, but 2, 4-dichlorophenoxyacetic acid had no effect in vitro. Chromatin from plants previously treated with 2, 4-dichlorophenoxyacetic acid supported a greater level of DNA synthesis than did chromatin from untreated plants.
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PMID:Enhanced deoxyribonucleic Acid polymerase activity of chromatin from soybean hypocotyls treated with 2,4-dichlorophenoxyacetic Acid. 1665 30


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